Studies On Editing Of Two Genes In Gibberellin Biosynthesis Pathway In Brassica Napus L.

Plant height is an important agronomic trait for yield trait formation of rapeseed.Research on plant height is beneficial to breed variety with elevated yield and lodging resistance.GA20ox and GA3ox are key enzymes in functional gibberellin biosynthesis.Present works aimed at enhancement of dwarf germplasm in rapeseed with gene editing technology,and generated dwarf materials which carry edited Bna GA20ox1 and Bna GA3ox1.The main results are as follows:1.Editing of gibberellin oxidase gene Bna GA3ox1（1）Gene cloning:Four homologous genes of Bna GA3ox1 were cloned from B.napus cv Nannong 9808.Sequences analysis showed that the four homologous Bna GA3ox1 are highly similar,and all of them contain 2OG-FeⅡ＿Oxy and Diox-N domains.（2）Gene expression:Bna GA3ox1 expression in different tissues of Nannong 9808 were determined,and results showed that Bna GA3ox1 was highly expressed in stem and leaf,implying that they may probably be involved in palnt stature development.Subcellular localization experiments with fusion vectors 35S:Bna A06g10250D:GFP showed that the homologs of Bna GA3ox1 are localized in the cytoplasm,cell membrane,and nucleus.（3）Gene editing:Using CRISPR/Cas9 technology,a double-target editing vector CRISPR-GA3 was constructed targeting four homologous genes of Bna GA3ox1.Transformation with the gene editing vector into Nannong 9808 generated four transgenic T₁plants.（4）Dwarf germplasm obtained:Dwarf plants were found in the T₂ generations derived from the CRISPR-GA3 vector.Detection of gene editing indicated editing events occurred at target sites of Bna GA3ox1.（5）Determination of gibberellin content:The gibberellin content in stem of the Bna GA3ox1 mutants with dwarf stature and the wild type control Nannong 9808 were determined.Results showed that the gibberellin content in stem of the dwarf mutants was lower than the control,which decreased by 20.2%.2.Editing of gibberellin oxidase gene Bna GA20ox1（1）Gene cloning:Four homologous genes of Bna GA20ox1 were cloned from B.napus cv Nannong 9808.Sequences analysis showed that the four homologous Bna GA20ox1 are highly similar,and all of them contain 2OG-FeⅡ＿Oxy and Diox-N domains.（2）Gene expression:Bna GA20ox1 expression in different tissues of Nannong 9808were determined,and results showed that Bna GA20ox1 was highly expressed in stem and leaf,implying that they may probably be involved in stem growth and leaf development.Subcellular localization experiments with fusion vectors 35S:Bna C01g17380D:GFP showed that the homologs of Bna GA20ox1 are localized in the cytoplasm,cell membrane,and nucleus.（3）Gene editing:Using CRISPR/Cas9 technology,a double-target editing vector CRISPR-GA20 was constructed targeting four homologous genes of Bna GA20ox1.Transformation with the gene editing vector into Nannong 9808,four transgenic plants were obtained.（4）Dwarf germplasm obtained:Dwarf plants were found in the T₂ generations derived from the CRISPR-GA20 vector.These results imply that our gene works have generated novel dwarf mutants useful for rapeseed breeding researches.